PROJECT SUMMARY The immunosuppressive tumor microenvironment creates a formidable challenge to treatment of pancreatic ductal adenocarcinoma (PDAC). Its resistance to the ?gold standard? chemo- and radiation therapy and to immune checkpoint blockade, which has recently been FDA approved for other cancer types, is motivation for to develop new therapies for treating PDAC. Poor responses to therapy are thought to be due to paucity of intratumoral effector T cells, which correlates with profound immunosuppression, insufficient antigen presentation as well as desmoplasia-driven compression of tumor vasculature. Recently, non-invasive focused ultrasound (FUS) has emerged as an immunomodulatory cancer therapy, with several studies showing primary and metastatic tumor burden reduction after ultrasound treatment. However, lack of knowledge regarding mechanisms of action with this very new technology as well as optimal ultrasound parameters to achieve consistently effective immunotherapy improvement presents a barrier to clinical or even widespread preclinical use. Furthermore, technical difficulties associated with imaging and consequently, precise treatment of pancreatic tumors with ultrasound, have complicated preclinical studies. Here, we propose a rigorous study of immunological biomarker changes in response to varying focused ultrasound parameters to both elucidate optimal ultrasound parameters as well as mechanism. Therapeutic ultrasound will be varied across ablative, mild hyperthermia, low-intensity radiation force, and cavitation regimes, with and without microbubbles. Our proposed research will use clinically meaningful orthotopic murine models of pancreatic cancer as well as multiplex mass cytometry-based immuno-phenotyping. Furthermore, we will use novel real-time high-resolution image-guided therapeutic FUS technology to enable precise image guided treatment. Once we have evaluated promising acoustic parameters, we will test the treatment protocols with immune checkpoint blockade to evaluate increase in therapeutic efficacy. This effort will inform the optimal design of combined ultrasound-immunotherapy strategies against PDAC.